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WO2014027211A1 - Complexes biomoléculaires - Google Patents

Complexes biomoléculaires Download PDF

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Publication number
WO2014027211A1
WO2014027211A1 PCT/GB2013/052191 GB2013052191W WO2014027211A1 WO 2014027211 A1 WO2014027211 A1 WO 2014027211A1 GB 2013052191 W GB2013052191 W GB 2013052191W WO 2014027211 A1 WO2014027211 A1 WO 2014027211A1
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WIPO (PCT)
Prior art keywords
labelling
biomolecular complex
biomolecular
residues
sequences
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PCT/GB2013/052191
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English (en)
Inventor
Philip Blower
Gregory Mullen
Jennifer Williams
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Cancer Research Technology Limited
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Publication of WO2014027211A1 publication Critical patent/WO2014027211A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1045—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants
    • A61K51/1072—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants the tumor cell being from the reproductive system, e.g. ovaria, uterus, testes or prostate

Definitions

  • the present invention relates to biomolecular complexes.
  • the biomolecular complexes of the invention are useful in molecular imaging, diagnostic and therapeutic applications.
  • the invention also relates to nucleic acid molecules encoding such biomolecular complexes, methods of producing such biomolecular complexes; biomolecular complexes for use as medicaments in radiotherapy; and methods of treatment, diagnosis and molecular imaging utilising such molecular complexes.
  • Radiopharmaceuticals are agents that may be used in a wide range of applications, including diagnostic, therapeutic or research uses.
  • Examples of radiopharmaceuticals include molecular imaging agents in which the detectable signal used in imaging is provided by a radionuclide that emits a gamma photon or a positron and which is attached or incorporated into a molecular or particulate entity which endows it with an affinity for a specific molecular target which is present in vivo.
  • the target molecule or pathway or physiological state may be one that acts as a biomarker for a specific physiological, biochemical, metabolic or gene expression phenotype such as may be present in disease sites (such as tumours or inflamed tissues).
  • the same molecular targeting principle can be used therapeutically by use of a particle- emitting radioisotope in place of the gamma or positron emitter.
  • the aim is to target the radioisotope to a location where emitted particles (e.g. alpha particles, beta particles, Auger electrons) can kill cells associated with the disease site.
  • emitted particles e.g. alpha particles, beta particles, Auger electrons
  • the targeting vehicle is often a protein (e.g. a monoclonal antibody, antibody fragment, signalling protein etc.) or peptide with specific affinity for a receptor, transporter or other molecular target.
  • these protein or peptide vehicles must be modified in order to incorporate a suitable radionuclide.
  • the modification may consist of direct attachment of a radionuclide, for example the substitution of a hydrogen in the phenolic group of a tyrosine residue with a radioactive iodine.
  • the radioisotope is a metal (e.g. Tc-99m, ln-1 11 , Cu-64, Ga-68 etc. for imaging and Re-188, Y- 90, Lu-177 etc.
  • Tc-99m is a preferred radioisotope for imaging because of its favourable gamma energy and ready availability from the Mo-99/Tc-99m generator.
  • Re-188 is an attractive therapeutic radioisotope because it too is available from a generator (W- 188/Re-188) and it is chemically analogous to Tc-99m.
  • the bioconjugate and the process that produces it will preferably satisfy a number of requirements, which may include some of the following:
  • the molecular recognition function of the protein or peptide must be preserved. This means the modification should be at a site on the molecule that is well-defined and remote from the target-binding site.
  • the amount of vehicle should be kept to a minimum whilst still incorporating the amount of radioactivity needed for the imaging procedure (i.e. the specific activity of the labelled product should be as high as possible), both to reduce the chances of producing a toxic or undesired physiological effect, for example saturation of the molecular target and thus altering the biological property it is intended to measure, and to reduce costs.
  • the labelling process must be as quick and simple as possible in order to reduce losses of radioactivity by decay, to reduce the opportunity for introduction of microbial contamination, and to reduce the radiation exposure of the operator.
  • the labelling yield should be >95% to avoid the necessity of incorporating additional purification steps to remove non-bound radioactivity
  • the product should as far as possible be homogeneous, that is every radiolabeled molecule should have the same structure, stability and affinity for the target
  • the product should be stable in vivo for the requisite period to perform the imaging procedure
  • a labelled biomolecular complex comprising the protein of interest, and a labelling sequence that mediates attachment to the chosen label
  • Fast labelling in which only a short incubation time is required for a label to become attached to the biomolecular complex (e.g. by conjugation via the labelling sequence) is highly beneficial.
  • Bifunctional chelators are molecules that comprise two functional components: a metal-binding moiety that is designed for fast binding of the radiometal combined with slow dissociation of the complex; and a biomolecule-binding moiety, such as an active ester that reacts with amine groups of lysine side chains of the biomolecule.
  • the challenge for achieving site-specific attachment of radiolabels is that the amino acid side chains with which these bifunctional chelators react are rarely unique in the biomolecule. For example, in proteins with multiple lysine residues, it is difficult or impossible to control the bioconjugation conditions to achieve modification at only one chosen site.
  • the radioisotope Tc-99m is preferred for reasons identified above. Its periodic congener rhenium can be used analogously by virtue of the availability of beta-emitting radioisotopes, Re-186 and Re-188. These radiometals have complex chemistry with a variety of ligand preferences and structures in different metal oxidation states. Two particularly stable complex types, or “cores”, have been widely used by virtue of their stability and synthetic accessibility from pertechnetate and perrhenate, the forms in which the radiometals are most readily available. These are the M0 3+ core, with the metal in oxidation state +5, and the M(CO) 3 + core, with the metal in oxidation state +1 (where M may, for example, be Tc or Re).
  • cores can be chelated by various chelating agents, such as the chelator-derivatised amino acids referred to above, which may be optimally designed for the particular core.
  • chelating agents such as the chelator-derivatised amino acids referred to above, which may be optimally designed for the particular core.
  • the requirements of the chelator design are different because of the different geometries preferred by the cores: typically the chelator should form the base of a square pyramidal complex for the M0 3+ core and the trigonal face of an octahedral complex for the M(CO) 3 + core.
  • Other cores can be considered but have not been exploited to a significant degree hitherto.
  • M0 3+ is particularly effective in the context of amino acid sequences containing a cysteine thiol.
  • the coordinating atoms in this context are believe to comprise typically the thiolate sulphur and three sequential anionic, deprotonated amide nitrogens of the peptide backbone.
  • Others have found that binding of Tc0 3+ , generated in situ by the reduction of pertechnetate with stannous compounds, to peptides is enhanced by the presences of a series of arginine residues. The mechanism of this enhancement is unclear.
  • the M(CO) 3 + core is particularly effective in the context of proteins containing a his-tag.
  • the coordinating ligands in this setting are believed to comprise two histidine imidazole groups and a third, as yet unknown, ligand.
  • This core is especially attractive for several reasons. It is particularly inert towards ligand substitution and oxidation and hence proteins labelled in this way show excellent in vivo stability.
  • the his-tag is a very common motif in recombinant proteins because of its value in protein purification.
  • the radioactive M(CO) 3 + synthon (often assumed to be [M(CO)3(H 2 0) 3 ] + ) is easily synthesised from MO 4 " by a simple kit-based method.
  • the radiolabelling works well and satisfies to a variable extent the six requirements set out above.
  • others require higher protein concentration or higher temperature reaction conditions and/or a subsequent purification step.
  • the presence of a his-tag does not in itself provide the desirable attribute that radiolabelling efficiency should be essentially independent of the particular protein.
  • the factors that dictate the labelling efficiency are unknown but must arise from features of the amino acid sequence in the vicinity of the his-tag. In the absence of a his-tag some proteins can still be labelled albeit with reduced efficiency, therefore it is not certain that the his-tag guarantees site -specificity.
  • a biomolecular complex comprising a protein of interest and a labelling sequence capable of conjugation to a metal tricarbonyl ([M(CO) 3 ] + ), wherein the labelling sequence consists of between 6 and 15 amino acid residues of which between 4 and 6 are histidine residues and
  • the labelling sequence has an isoelectric point (pi) of at least 9.
  • nucleic acid molecule encoding a biomolecular complex in accordance with the first aspect of the invention.
  • a method of manufacturing a biomolecular complex according to the first aspect of the invention comprising expressing a nucleic acid according to the second aspect of the invention to yield a biomolecular complex.
  • a biomolecular complex according to the first aspect of the invention and further comprising a conjugated metal tricarbonyl comprising a radionuclide, for use as a medicament in radiotherapy.
  • a method of treatment comprising providing a therapeutically effective amount of a biomolecular complex according to the first aspect of the invention, further comprising a conjugated metal tricarbonyl comprising a radionuclide, to a subject in need thereof.
  • Suitable methods of treatment in accordance with this aspect of the invention are described elsewhere in the present application.
  • such methods of treatment may make use of biomolecular complexes in which the protein of interest is one that associates with a marker associated with the disease to be treated, thus targeting the radionuclide (associated with the tricarbonyl) to a site where it can achieve a therapeutic impact upon cells associated with the disease to be treated.
  • the studies reported in more detail below illustrate that the biomolecular complexes of the invention are capable of incorporating antibodies such as those directed to prostate specific membrane antigen (PSMA), a marker expression of which is frequently up-regulated at sites of prostate cancer.
  • PSMA prostate specific membrane antigen
  • a method of molecular imaging comprising providing a biomolecular complex according to the first aspect of the invention, further comprising a conjugated metal tricarbonyl comprising, to a subject, and determining the location of the conjugated metal tricarbonyl within the subject, wherein the location of the conjugated metal tricarbonyl within the subject is indicative of the location of a binding partner of the protein of interest.
  • biomolecular complexes of the invention are also suitable for use in a number of screening applications.
  • biomolecular complexes of the invention may be used to identify proteins of interest capable of interacting with a desired binding partner.
  • multiple biomolecular complexes incorporating different proteins of interest may be produced, for example, by means of expression libraries in which the proteins of interest are associated with a suitable labelling sequence (thus effectively giving rise to libraries of biomolecular complexes of the invention).
  • a plurality of biomolecular complexes, comprising a plurality of proteins of interest may then be provided to a sample comprising the desired binding partner (such as a cell expressing a molecule to which it is desired to identify a protein of interest that can be used for cellular targeting).
  • the presence of a protein of interest capable of binding to the desired binding partner can be identified by localisation of the metal tricarbonyl label with the sample. Further analysis may then be undertaken to identify which of the proteins of interest within the plurality of biomolecular complexes is responsible for this labelling (and thus which protein of interest interacts with the binding partner in question).
  • biomolecular complexes of the invention may make use of a plurality of biomolecular complexes comprising a plurality of proteins of interest, wherein the proteins of interest have been selected for their ability to interact with a desired binding partner.
  • the plurality of proteins of interest may comprise different antibody fragments or derivatives (e.g. SFv, diabodies, or minibodies) each directed to the same antigen.
  • the protein of interest having most favourable binding characteristics can then be identified by assessing which of the biomolecular complexes exhibits the best binding, and then analysing this biomolecular complex to identify which of the proteins of interest it contains.
  • biomolecular complexes of the invention are able to prove beneficial in these various important clinical and research applications.
  • Figure 1 shows the results of a study investigating radiochemical yield of the His/Cys Tag peptide on the CelluspotTM peptide array
  • Figure 2 shows the results of a study investigating radiochemical yield of the His/Cys Tag peptide in solution
  • Figure 3 shows the results of a study investigating the correlation between the radiochemical yield of the His/Cys Tag peptides in solution and on solid phase
  • Figure 4 shows the results of a study investigating relative radiochemical yields of all 384 peptides on the CelluspotTM array post labelling with [99mTc(CO)3]+. Results obtained after 30 minutes of labelling in PBS buffer at pH 7.4. The peptides have been categorised according to their main characteristics. All peptides other than the controls contain at least 1 histidine residue
  • Figure 5 shows the results of a comparison study between the labelling of peptide sequence with multiple negatively charged amino acids and positively charged amino acids.
  • Sequences include at least 2 negatively charged amino acids (glutamic acid and/or aspartic acid) or 2 positively charged amino acids (arginine and/or lysine)
  • Figure 6 shows the results of a study investigating HHHCHHHXLAAAL Sequences where X differs between each sequence.
  • the amino acids varied include positively charged Arg and Lys, negatively charged Glu, Asp and neutral Gly
  • Figure 7 shows the results of a comparison study between Glutamic acid and Arginine containing sequences. A single amino acid has been changed within the sequences
  • Figure 8 shows the results of a comparison study between R, G, S, E and D containing sequences. A single amino acid is replaced in the same position within each sequence
  • Figure 9 shows the results of a study investigating His/Cys Tag sequences in which a single amino acid has been replaced.
  • His/Cys Tag sequence In the original His/Cys Tag sequence a glutamic acid residue was included next to the histidines. This has been changed from E to D, S, K, R or no amino acid. The influence of charged amino acids on the labelling of histidines can be easily observed
  • Figure " ! 0 shows the results of a study investigating radiochemical yield plotted against isoelectric point for all peptide sequences with a His-Tag i.e. 6 consecutive histidines HHHHHH
  • Figure 1 1 shows the results of a study investigating radiochemical yield plotted against isoelectric point for all comparator peptide sequences with 3 histidines HXHXHX
  • Figure 12 shows the results of a study investigating radiochemical yield plotted against isoelectric point for all peptide sequences with 4 consecutive histidines HHHH
  • Figure13 shows the results of a study investigating radiochemical yield plotted against isoelectric point for all comparator peptide sequences with 2 histidines HXHX
  • Figure 14 shows the results of a study investigating radiochemical yield plotted against isoelectric point for all comparator peptide sequences with 2 histidines HXXH
  • Figure 15 shows the results of a study investigating number of arginine residues plotted against radiochemical Yield and isoelectric point of sequences that contain 6 consecutive histidines (a His-Tag). Optimum number of arginines appears to be between 2-4 arginine residues within a sequence. There is a significant difference (p ⁇ 0.0001 ) between the radiochemical yield for 1 arginine and 2 arginines. The pi and the radiochemical yield of the peptide sequences increase in proportion to the number of arginines present in the sequence.
  • Figure 16 shows the results of a study investigating number of lysine residues in a peptide sequence that contains 6 consecutive histidines (a His-Tag) plotted against isoelectric point and radiochemical yield.
  • the results are very similar to that of arginine however, lysine has a side chain with a lower pKa therefore more lysines are needed to give an equivalent pi to sequences including arginines.
  • This is shown in the top graph where a significant difference in radiochemical yield is observed between sequences with 2 and three lysine residues. Consequently, 3-5 lysine residues are favoured.
  • pi and radiochemical yield of the peptide sequences are proportional to the number of lysine residues present.
  • Figure 17 shows the results of a study investigating combined arginine and lysine containing peptide sequences plotted against pi and radiochemical yield. This highlights the fact that arginine is a more positively charged amino acid and can have a greater influence on the increase in pi of a peptide sequence. Consequently it also has a greater influence on the radiochemical yield.
  • the binding affinities of the [M(CO) 3 ] + complexes to histidines adjacent to multiple arginine residues is greater than to histidines adjacent to multiple lysine residues.
  • a larger number of lysines are required in the labelling sequence to provide the same labelling ability of the arginine containing sequences.
  • Figure 18 shows the results of a comparison study between different positions of the arginine residues in the labelling sequences with respect to the histidines.
  • FIG. 20 shows the results of a study investigating radiolabelling efficiencies with [ 99m Tc(CO) 3 ] + high-throughput screening methodology for His-tag peptide sequences of the invention compared with prior art labelling sequences and comparator sequences.
  • Figure 21 shows the results of a study comparing radiochemical yield of the top 10 sequences with and without a cysteine residue
  • Figure 22 shows the results of a study investigating radiochemical yield for identical peptide sequences in which a cysteine has been replaced with methionine.
  • the difference in labelling sequences containing a methionine rather than cysteine is insignificant. Consequently, methionines can be used instead of cysteine to give an almost identical rate of labelling while avoiding disulfide bond formation.
  • a few comparisons have been made between the cysteine/methionine containing sequences and those without. There is a difference in radiochemical yield when the Met and Cys are removed
  • Figure 23 shows the results of a study investigating radiochemical yield of all peptide sequences within the assigned categories. All peptides that include an arginine or multiple lysine have been highlighted. This demonstrates that the sequences with the highest radiochemical yield in each category always contain either an arginine or lysine.
  • FIG. 24a SDS-PAGE and Western Blot of the extraction and purification process of the J591 scFvJWT protein from the culture supernatant. Lanes in both left and right hand images are as follows: A is culture supernatant; B is 35mM imidazole wash of the NiNTA column; C is J591 scFvJWT elution from NiNTA column (250mM imidazole); D is SEC Purification: Fraction 1 - BSA Protein; E is SEC Purification: Fraction 2 - Non-covalent dimers of J591 scFvJWT; F is SEC Purification: Fraction 3 - Purified monomeric J591 scFvJWT; and G is concentrated purified J591 scFvJWT - 1 .3mg/ml.
  • FIG. 24b SDS-PAGE and Western Blot of the extraction and purification process of the J591 scFv protein from the culture supernatant. Lanes in both left and right hand images are as follows: A is culture supernatant; B is 35mM imidazole wash of the NiNTA column; C is J591 scFv elution from NiNTA column (250mM imidazole); D is SEC Purification: Fraction 1 - BSA Protein; E is SEC Purification: Fraction 2 - Non-covalent dimers of J591 scFv; and F is SEC Purification: Fraction 3 - Purified monomeric J591 scFv. Left: SDS-PAGE with rows from A-F. Right: Western Blot of the SDS-PAGE with lanes from A-F. J591 scFv sample runs as a single band corresponding to the size of the monomer, 27kDa.
  • FIG. 25 HPLC SEC analysis of the scFv proteins shows elution primarily as a single monomeric species at 9min. The peak at 8min represents the non-covalent dimers of the scFv protein.
  • FIG. 26 Radiolabelling efficiencies of the J591 scFvJWT, J591 scFv, huJ591 , 6C7.1 and 6C7.1 -Cys proteins under increasingly dilute conditions expressed as % radiochemical yield against log[protein].
  • Table 4 reveals the concentration in uM and mg/ml corresponding to the log[protein] data points on the graphs. The labelling efficiency was recorded at 5 different time points: Panel A) 15 minutes, Panel B) 30 minutes, Panel C) 60 minutes, Panel D) 90 minutes and Panel E) 120 minutes.
  • FIG. 29 Serum stability of [ 99m Tc(CO) 3 ] + -J591 (scFv)JWT by SDS-PAGE and Coomassie staining (A) and autoradiograph (B) for the serum stability analysis of J591 scFvJWT for 4 hours at 37°C. Lanes are as follows: A is serum proteins + [ 99m Tc(CO) 3 ] + ; B is [ 99m Tc(CO) 3 ] + -
  • Table 1 shows a number of sequences suitable for use as labelling sequences in the biomolecular complexes of the invention (as well as a number of comparator sequences),
  • Table 2 shows a subset of these sequences.
  • Table 3 sets out the amino acid sequences of the scFv protein fragments: J591 scFvJWT, J591 scFv, huJ591 , 6C7.1 scFv, 6C7.1 CscFv referred to in Study 2. Labelling sequences have been highlighted in bold.
  • Table 4 sets out details of the protein concentrations used in Study 2 and shown in Figure 26.
  • Table 5 sets out details of protein concentrations and incubation times at which J591 (scFv)JWT demonstrates a radiochemical yield greater than or equal to 95%.
  • the biomolecular complexes of the invention comprise a protein of interest and a labelling sequence. These labelling sequences allow the biomolecular complexes to be conjugated with metal tricarbonyl.
  • the biomolecular complexes of the invention may further comprise a metal tricarbonyl conjugated to the labelling sequence. Further details of such embodiments are described in more detail elsewhere in the specification.
  • the metal tricarbonyls are able to serve as labels that can subsequently be detected, for example by detection of a radionuclide incorporated in the metal tricarbonyl, or by infrared- based techniques.
  • a biomolecular complex of the invention may comprise a labelling sequence having a pi of at least 9.5, at least 10, or even at least 10.5.
  • references to the isoelectric point should be taken as being the isoelectric point as determined using compute pl/Mw by ExPASy Bioinformatics Resource Portal.
  • Compute pl/Mw is a tool which allows the computation of the theoretic pi (isoelectric point) and Mw (molecular weight) for a list of UniProt Knowledgebase (Swiss Prot or TrEMBL) entries or for user entered sequences.
  • the biomolecular complexes of the invention comprise labelling sequences having a length of between 6 and 15 amino acid residues.
  • a suitable biomolecular complex may comprise a labelling sequence consisting of 6, 7, 8, 9, 10, 1 1 , 12, 13, 14 or 15 amino acid residues.
  • the labelling sequence consists of between 12 and 15 amino acid residues.
  • biomolecular complexes of the invention comprise between 4 and 6 histidine residues.
  • suitable embodiments include those wherein 4, 5 or 6 histidine residues are present.
  • a suitable embodiment comprises the 6 residues arranged as two groups of three contiguous histidine residues, the two groups being separated by a single non-histidine residue.
  • the 6 histidine residues are contiguous with one another.
  • Biomolecular complexes of the invention may comprise a labelling sequence wherein the histidine residues are located at an end of the labelling sequence.
  • an end should be construed as requiring that the histidine residues provide the initial or final amino acids of the labelling sequence.
  • the histidine residues may be provided solely at an end of the labelling sequence. In other embodiments one or more of the histidine residues are provided at an end of the labelling sequence, and further histidine residues are provided elsewhere in the labelling sequence.
  • biomolecular complexes of the invention all of the histidine residues are embedded within the labelling sequence.
  • reference to amino acid residues being "embedded within" a sequence should be construed as requiring that the residues referred to are not present at an end of the sequence.
  • Biomolecular complexes of the invention comprise labelling sequences in which at least 2 of the amino acid residues present are selected from the group consisting of lysine and/or arginine residues. Accordingly, it will be recognised that in suitable embodiments the biomolecular complexes of the invention may make use of labelling sequences that comprise a single lysine residue and a single arginine residue. In suitable embodiments the biomolecular complexes of the invention may make use of labelling sequences that comprise two or more lysine residues. In suitable embodiments the biomolecular complexes of the invention may make use of labelling sequences that comprise two or more arginine residues.
  • Biomolecular complexes of the invention may utilise labelling sequences in which both lysine and arginine residues are present.
  • suitable biomolecular complexes include those that comprise labelling sequences in which lysine residues are present, but arginine residues are absent.
  • suitable biomolecular complexes of the invention include those that comprise labelling sequences in which arginine residues are present, but lysine residues are absent.
  • biomolecular complexes of the invention may comprise a labelling sequence comprising at least 3 lysine residues.
  • the biomolecular complexes of the invention may make use of a labelling sequence that comprises between 3 and 5 lysine residues.
  • a biomolecular complex of the invention may incorporate a labelling sequence that comprises 3 or 4 lysine residues, such as a labelling sequence in which 4 lysine residues are present.
  • the biomolecular complexes of the invention may include a labelling sequence that comprises between 2 and 5 arginine residues.
  • a suitable biomolecular complex of the invention may include a labelling sequence that comprises between 2 and 4 arginine residues, such as a labelling sequence in which 3 arginine residues are present.
  • biomolecular complexes of the invention may comprise a labelling sequence that does not contain aspartic acid residues.
  • biomolecular complexes of the invention may comprise a labelling sequence that does not contain glutamic acid residues.
  • the biomolecular complexes of the invention may comprise a labelling sequence that contains neither aspartic acid nor glutamic acid residues.
  • the biomolecular complexes of the invention may include a labelling sequence that comprises a cysteine or methionine residue.
  • a cysteine or methionine residue in accordance with this embodiment may be preferred in examples of biomolecular complexes comprising one or more arginine residues.
  • biomolecular complexes comprising labelling sequences that comprise arginine residues in combination with a methionine residue constitute preferred embodiments of the present invention.
  • Cysteine residues have been shown to improve the conjugation of histidine tagged peptides (such as labelling sequences) with metal tricarbonyls.
  • the inventors have surprisingly found that similar advantages are also provided by the provision of one or more methionine residues within the labelling sequence.
  • cysteine is present in the labelling sequence
  • a biomolecular complex of the invention may utilise a labelling sequence that does not contain cysteine.
  • the biomolecular complexes of the invention optionally comprise a metal tricarbonyl.
  • a metal tricarbonyl core, conjugated to the labelling sequence forms part of the biomolecular complex.
  • the metal tricarbonyl may comprise a complex or fragment wherein the metal has a d 10 electron configuration (e.g. Tc(l), Re(l), Cr(0), Mo(0), W(0)) and three carbonyl ligands coordinated mutually cis to each other (i.e. facially).
  • a d 10 electron configuration e.g. Tc(l), Re(l), Cr(0), Mo(0), W(0)
  • three carbonyl ligands coordinated mutually cis to each other (i.e. facially).
  • the metal tricarbonyl is selected from the group consisting of: [Mo(CO) 3 ]; [Cr(CO) 3 ]; [W(CO) 3 ]; [Tc(CO) 3 ] + ; and [Re(CO) 3 ] + .
  • the biomolecular complexes of the invention may include a metal tricarbonyl that comprises a radionuclide. Such complexes may be of use in applications such as radiotherapy (particularly targeted radionuclide therapy), or radioimaging, as discussed elsewhere in the specification.
  • the radionuclide may comprise technetium, and the technetium may be selected from the group consisting of: Tc-99; Tc-99m; and Tc-94m.
  • the radionuclide may comprise rhenium, and the rhenium may be selected from the group consisting of: Re-188; and Re-186.
  • biomolecular complexes in accordance with the present invention may make use of a non-radioactive metal tricarbonyl comprising rhenium, chromium, molybdenum or tungsten.
  • a suitable metal tricarbonyl for use in such embodiments may comprise rhenium selected from the group consisting of: Re-185; and Re-187.
  • the protein of interest incorporated in biomolecular complexes of the invention will generally be selected with reference to a binding partner (i.e. another molecule, such as a protein or other biological molecule) with which the protein of interest interacts.
  • a binding partner i.e. another molecule, such as a protein or other biological molecule
  • the protein of interest can be used to target the metal tricarbonyl (attached to the protein of interest via the labelling sequence) to the location of the binding partner.
  • Metal tricarbonyls are detectable via infrared techniques.
  • biomolecular complexes of the invention incorporating metal tricarbonyls are suitable for use in techniques where the presence (and optionally location) of the metal tricarbonyl is determined via infrared.
  • these may include such techniques as infrared surface enhanced spectroscopy,
  • Metal tricarbonyls comprising a radionuclide are readily detected (by virtue of their emission of radiation), and thus represent useful agents for use in molecular imaging applications in which it is desired to determine the location of a molecule (which is a binding partner for a protein of interest) within a sample.
  • Biomolecular complexes comprising a metal tricarbonyl with a radionuclide that is a gamma radiation or positron emitter are particularly suitable for use in such embodiments.
  • metal tricarbonyls comprising a radionuclide that is a gamma radiation or positron emitter are well suited for use in embodiments of such aspects in which the location of the metal tricarbonyl is to be determined by detection of radioactivity.
  • the biomolecular complexes of the invention are also suitable for use in therapeutic roles. These include use as medicaments for radiotherapy, which may be used in applications such as the methods of treatment of the fifth aspect of the invention.
  • the protein of interest may be selected so that it has a binding partner associated with a disease condition.
  • the binding partner may be a molecule associated with cancer, such as a marker expressed by cancer cells.
  • the interaction of binding partner with the protein of interest can thus target the biomolecular complex, to the site of the disease.
  • the biomolecular complex may comprise a metal tricarbonyl incorporating a radionuclide that is able to kill cells associated with the disease condition, such as cancer cells.
  • Radionuclides suitable for use in such embodiments include particle emitters, and it will be appreciated that such radionuclides represent preferred components that may be utilised in metal tricarbonyls utilised in biomolecular complexes for medical use, such as methods of treatment.
  • the protein of interest to be utilised in a biomolecular complex of the invention may be an antibody or antibody fragment, and the binding partner may be the antigen recognised by said antibody or antibody fragment (such as an scFv antibody).
  • results set out elsewhere in the specification illustrate the suitability of antibodies (or antibody fragments) to serve as proteins of interest in the biomolecular complexes of the invention.
  • This utility is illustrated with (but not limited to) biomolecular complexes comprising the anti-prostate specific membrane antigen (PSMA) antibody J591 , and in particular scFv antibodies derived from this.
  • PSMA anti-prostate specific membrane antigen
  • the protein of interest may be an enzyme, and the binding partner may be a substrate of said enzyme.
  • the protein of interest may be a substrate of an enzyme, and the binding partner may be said enzyme.
  • the protein of interest and binding partner may be selected from a ligand and receptor known to bind to one another.
  • suitable proteins of interest include: neurotransmitters; hormones; albumin; and amyloid precursors.
  • suitable proteins of interest include synthetic proteins, which may be designed to bind to a desired target.
  • Suitable biomolecular complexes in accordance with the present invention include those comprising labelling sequences selected from the group consisting of: HHHHHHALRRRLC HHHHHHALRRRLM; CLRRRLAHHHHHH; MLRRRLAHHHHHH; HHHHHHALRRRLKC HHHHHHALRRRLKM; CKLRRRLAHHHHHH; MKLRRRLAHHHHHH; CRRHHHHHHRRC MRRHHHHHHRRM; GRRHHHHHHRRG; HHHHHHRRAARRC; HHHHHHRRAARRM CRRAARRHHHHHH; MRRAARRHHHHHH; HHHHALRRRL; LRRRLAHHHHHH RCRGHHHHHHGRCR; RMRGHHHHHHGRMR; GGRHHHRHHHRGG
  • HHHHHHRGGGRC HHHHHHRGGGRM
  • CRGGGRHHHHHH MRGGGRHHHHHH HHHHHHRARARC
  • HHHHHHRARARM CRARARHHHHHH; MRARARHHHHHH GKKHHHHHHKKG; HHHHHHRARAR; RARARHHHHHH; HHHHGRGGRC HHHHHHGRGGRM; CRGGRGHHHHHH; GKKHHHHHHKKGC GKKHHHHHHKKGM; CGKKHHHHHHKKG; MGKKHHHHHHKKG; HHHHRRAARR RRAARRHHHHHH; HHHHHHRGGGR; RGGGRHHHHHH; HHHHKGGGK KGGGKHHHHHH; HHHHHHKAKAK; and KAKAKHHHHHH.
  • biomolecular complexes in accordance with the present invention include those comprising labelling sequences selected from the group consisting of: HHHHHHALRRRLC; HHHHHHALRRRLM; CLRRRLAHHHHHH; MLRRRLAHHHHHH; HHHHHHALRRRLKC; HHHHHHALRRRLKM; CKLRRRLAHHHHHH; MKLRRRLAHHHHHH; CRRHHHHHHRRC; MRRHHHHHHRRM; GRRHHHHHHRRG; HHHHHHRRAARRC; HHHHHHRRAARRM; CRRAARRHHHHHH; MRRAARRHHHHHHALRRRL; LRRRLAHHHHHH; RCRGHHHHHHGRCR; RMRGHHHHHHGRMR; GGRHHHHHHRGG; HHHHHHRGGGRC; HHHHHHHH
  • biomolecular complexes of the invention include those comprising the labelling sequence LRRRLAHHHHHH, or CLRRRLAHHHHHH, or HHHHHHALRRRLC. Further suitable examples of preferred biomolecular complexes include those comprising the labelling sequence MLRRRLAHHHHHH or HHHHHHALRRRLM.
  • the invention also provides a nucleic acid molecule encoding a biomolecular complex in accordance with the first aspect of the invention, and the translation of such nucleic acids to yield a biomolecular complex represents a suitable method of manufacturing a biomolecular complex of the invention.
  • Such expression may be accomplished by cellular expression, or in vitro translation/transcription approach.
  • biomolecular complexes of the invention may also be produced by other means, including de novo synthesis. Suitable means of synthesis will be apparent to those skilled in the art.
  • Methods in accordance with the third aspect of the invention may optionally further comprise a step of conjugating the biomolecular complex (produced on expression of the nucleic acid) with a metal tricarbonyl (of any sort considered herein).
  • biomolecular complexes of the invention provide notable advantages in terms of their efficiency in conjugating with metal tricarbonyls. These advantages give rise to certain beneficial embodiments of the methods of the invention by which such conjugated biomolecular complexes of the invention can be manufactured. These methods may be used to achieve levels of conjugation of 95% or more, levels which are considered useful in both clinical and pre-clinical applications.
  • a method of manufacturing a biomolecular complex comprising a metal tricarbonyl (optionally comprising a radionuclide) as considered elsewhere in this disclosure may utilise a concentration of the biomolecular complex, at the time that the conjugation is performed, of between approximately 5 ⁇ and approximately 30 ⁇ .
  • the concentration of the biomolecular complex at conjugation is selected from the group consisting of: approximately 28 ⁇ ; approximately 14 ⁇ ; and approximately 7 ⁇ .
  • a method of the invention may utilise a concentration of the biomolecular complex (at the time that conjugation is performed) of below approximately 15 ⁇ , or even below, 10 ⁇ , 9 ⁇ , or 8 ⁇ .
  • concentration of the biomolecular complex at the time that conjugation is performed
  • the efficiency of labelling of the biomolecular complexes of the invention is so high that useful levels of metal tricarbonyl conjugated biomolecular complexes can even be achieved using concentrations of the biomolecular complexes as low as 7 ⁇ .
  • the efficiency of labelling of biomolecular complexes of the invention is so high that useful levels of conjugation may be achieved using only short periods of incubation between the biomolecular complexes and metal tricarbonyls.
  • rapid labelling methods of this sort provide considerable practical advantages.
  • conjugation may be conducted over a period of between about 25 and 130 minutes.
  • such a method may be one wherein the conjugation is conducted over a period of between about 30 and 120 minutes.
  • useful levels of tricarbonyl labelling may be achieved in methods in which the conjugation is conducted over a period of less than 90 minutes, for example a period of 60 minutes or less. Suitable useful levels of labelling may even be achieved using methods wherein the conjugation is conducted over a period of approximately 30 minutes.
  • the efficiency of labelling of biomolecular complexes of the invention is so high that the threshold labelling efficiency of 95% which typically would obviate the need for post- labelling purification, is reached at low protein concentrations in a short time. Elimination of post-labelling purification considerably simplifies the procedure.
  • the efficiency of labelling of the sequences of the invention in the biomolecular complexes is so high compared to other alternative labelling sequences known from the prior art (such as sequences containing one or two histidines) that such a high level of site- specificity can be achieved that the binding affinity of the protein of interest to its target should not be adversely affected.
  • biomolecular complexes of the invention conjugated to metal tricarbonyl groups, are readily able to achieve very high levels of labelling efficiency, at or above the 95% degree that is considered important for clinical or other biological uses.
  • These very high levels of labelling efficiency obviate the need for a purification step prior to use, which in turn provides substantial benefits in terms of the simplicity, quality, and speed of the labelling procedure.
  • Figure 1 shows the labelling efficiencies of the various labelling sequences when provided on solid phase arrays (i.e. conjugated to CelluspotTM peptide array), while Figure 2 shows labelling efficiency when the labelling sequences are provided as individual peptide chains in solution.
  • Figure 3 it is possible to make a direct correlation between the results achieved in solution and those achieved in the solid phase, illustrating that the model used is of high quality. This proves that labelling efficiencies observed in the studies set out below (in which labelling efficiency is investigated through analysis of [ 99m Tc(CO) 3 ] + labelled CelluspotTM arrays) can be considered an accurate reflection of the situation in solution.
  • the isoelectric point (pi) is the pH at which a protein carries no net charge. At pH values below the isoelectric point proteins carry a net positive charge, above it a net negative charge.
  • pi has been calculated using compute pl/Mw by ExPASy Bioinformatics Resource Portal.
  • Compute pl/Mw is a tool which allows the computation of the theoretic pi (isoelectric point) and Mw (molecular weight) for a list of UniProt Knowledgebase (Swiss Prot or TrEMBL) entries or for user entered sequences.
  • the presence of positively charged amino acids in a peptide sequence will increase the pi and peptides with high pi are also associated with high radiochemical yields.
  • Figures 10 to 14 set out 5 graphs in which pi is plotted against the radiochemical yields of peptide sequences that have
  • the inventors' studies further illustrated that the presence of positively charged amino acid residues in labelling sequences increases the ability of histidine residues within these sequences to conjugate metal tricarbonyls.
  • Figures 4 and 23 plot the radiochemical yield of a number of peptide sequences (comprising labelling sequences suitable for use in the biomolecular complexes of the invention, and comparator sequences) after 15 minutes of incubation with [ 99m Tc(CO) 3 ] + .
  • Figure 23 provides further information regarding the presence of lysine and/or arginine residues over all peptide sequence categories. The results achieved clearly highlight that the sequences that contain positively charged amino acids are the ones that have the highest rate of labelling.
  • Peptide sequences can be placed into categories based on the amino acids that they contain and the arrangement of the amino acids with regards to the histidines.
  • Figure 4 and Figure 23 demonstrate the importance of positively charged amino acids in a labelling sequence for conjugation with [M(CO) 3 ] + .
  • Figure 5 compares labelling of sequences comprising positively charged amino acid residues with those comprising negatively charged amino acid residues. There is a significant difference (p ⁇ 0.005) between the average labelling efficiency of the sequences containing negatively charged amino acids and the sequences containing positively charged amino acids.
  • Figures 6 to 9 demonstrate results of binding studies using sequences in which a single amino acid is changed in order to compare direct effects on the labelling of the histidines.
  • the amino acids that are varied include glutamic acid (E), aspartic acid (D), lysine (K), arginine (R), serine (S), glycine (G).
  • the sequences containing aspartic acid and glutamic acid have the lowest labelling efficiency due to the negatively charged amino acids.
  • Sequences containing lysine and arginine have the highest labelling efficiency due to the presence of positively charged amino acids
  • positively charged amino acids e.g. lysine and arginine
  • negatively charged amino acids e.g. aspartate and glutamate
  • the presence of positively charged amino acids in the labelling sequences of the invention provides significant benefits by improving the ability of such labelling sequences to bind metal tricarbonyls via their conjugation with histidine residues present in the labelling sequence.
  • the labelling sequences of biomolecular complexes of the invention must contain between 4 and 6 histidine residues, and at least 2 residues are selected from the group consisting of lysine and/or arginine residues. A study was undertaken to identify preferred arrangements of these requisite amino acid residues within the labelling sequences of biomolecular complexes of the invention.
  • cysteine residues While the potential benefits of incorporating cysteine residues in sequences for conjugation with metal tricarbonyls have previously been recognised, the inventors surprisingly found that similar benefits can be provided by the use of methionine and that methionine can be used as a replacement for cysteine (see results in Figure 22).
  • the use of methionine residues rather than cysteine residues may provide advantages, in that the methionine residues are not associated with the formation of disulphide bonds in biomolecular complexes of the invention.
  • the inventors have identified a particularly effective version of a labelling sequence that may be used in the biomolecular complexes of the invention. This has the amino acid sequence CLRRRLAHHHHHH (or HHHHHHALRRRLC if the histidine sequence is provided at the other terminus).
  • this preferred labelling sequence was compared with that of prior art sequences and with comparator sequences.
  • the results of this study are shown in Figure 20.
  • the present study shows that labelling sequences for use in the biomolecular complexes of the present invention (such as the preferred sequence CLRRRLAHHHHHH) are able to provide a degree of labelling that is has a significant 8 fold higher labelling efficiency at 15 minutes (p ⁇ 0.00001 ) than the prior art labelling sequence (CKLAAALEHHHHHH).
  • This prior art sequence had previously been identified as having beneficial labelling properties, and these results show that the biomolecular complexes of the invention, utilising labelling sequences as described herein, will provide practical advantages over such complexes of the prior art.
  • the plate is exposed to the phosphor film for 5 minutes and the film is placed in the phosphor imager to generate an image.
  • J591 single chain designated J591scFv
  • huJ591 scFv humanised version of the J591 scFv
  • Arg/His Arg containing His-Tag sequences as having the highest affinity for the [ 99m Tc(CO) 3 ] + . Based on these results, these sequences should provide an efficient method for the fast labelling of proteins at low concentrations.
  • JWT preferred Arg/His labelling sequence of the invention referred to in the study above (designated "JWT” for the purposes of this second study) was engineered into the J591 single chain antibody (scFv) at the C-terminal.
  • J591 (scFv) is an antibody derived from the monoclonal antibody J591 which recognises an extracellular epitope of prostate specific membrane antigen (PSMA).
  • PSMA is a well-established marker for prostate carcinoma with elevated expressions detected in virtually all prostate cancers. For hormone refractory and metastatic disease, a further increase in levels of PSMA expression is observed.
  • a deimmunised version of the imAb J591 has been characterised in a number of different clinical studies including: phase I combined radioimmunotherapy and imaging trials with 111 ln-J591/ 90 Y-J591 and 177 Lu-J591 and imaging studies with 111 ln labelled J591 . Excellent targeting properties were observed for the imAb J591 however, due to the use of a full length antibody conjugate a long circulation time was observed and images had to be performed days after the injection of the tracer in order to obtain a sufficient contrast.
  • J591 scFv An Arg/His sequence, LRRRLAHHHHHH (JWT), was engineered at the C-terminal of the J591 scFv antibody producing the J591 scFvJWT antibody fragment (a biomolecular complex in accordance with the present invention).
  • Alternative fragments derived from imAb J591 were also produced, to act as comparators for the biomolecular complex of the invention.
  • Both fragments, J591 scFv and huJ591 scFv contained C-terminal (His)6-Tags.
  • scFv fragments of an antibody directed against mouse vascular cell adhesion molecular 1 (VCAM-1 ) were obtained for further comparative experiments.
  • 6C7.1 and 6C7.1 -Cys contain a (His)6-Tag at the C-terminal and will determine whether the Arg/His system can demonstrate a superior labelling efficiency over any His-Tagged protein independently of the protein sequence.
  • a Cys residue is present at the C-terminal side of the His-Tag. This provides a direct comparison between the previously successful Cys/His-Tag system and the newly developed Arg/His-Tag combinations of the invention for labelling proteins with [ 99m Tc(CO) 3 ] + .
  • a single chain fragment variable (scFv) of J591 in VH-VL orientation was PCR amplified from the SFG P28z vector and subcloned into a hybrid expression vector based on Psectag2 (Life Technologies) and Pires-Egfp (clonetech) sequences.
  • J591 JWT was generated by insertion of annealed overlapping oligonucleotides (Integrated DNA Technologies) with Notl/EcoRI overhangs replacing the original (His)6 sequence in the expression vector.
  • the sequence coding for the 6C7.1 scFv was kindly provided by Prof S. Duebel, University of Braunscheig, Germany.
  • the 6C7.1 scFv sequence was PCR amplified from a source vector and subcloned into the target expression vector.
  • 6C7.1 scFv with a C-terminal cysteine (6C7.1 -Cy(scFv)) was used as an additional control. All sequences were verified by DNA sequencing.
  • HEK393T cells were transfected with the respective expression vector and transfected cells were selected with 100ug/ml of Zeocin. Cells were then expanded to triple layer flasks and culture supernatants containing the recombinant protein were collected.
  • the scFv antibodies were extracted from HEK293T culture supernatant using Ni-NTA chromatography with a 5ml or 1 ml Ni-NTA column (Superflow cartridge, Qiagen).
  • a gel filtration step (Superdex 75 HR 10/30) using the AKTA-FPLC further purified the antibody fragments separating the residual BSA protein and dimerised scFv fragments from the monomeric scFv.
  • the purified monomeric scFv proteins in PBS at pH 7.4 were concentrated using VivaSpin molecular weight cut off filter columns (Sartorius).
  • the proteins were concentrated to the following concentrations: 1 .3mg/ml for J591 scFvJWT, 1 .35mg/ml for J591 scFv, 1 .9mg/ml for huJ591 scFv, 1 mg/ml for 6C7.1 and 1 .1 mg/ml for 6C7.1 C.
  • Protein concentration was measured by UV spectrometry with a UV absorption of 280nm using a Nanodrop device.
  • a molar extinction coefficient and molecular weight of the respective protein was determined from the primary aminoacid sequence using the ProtParam online tool, assuming all cysteines are present as cystines: for J591 scFvJWT E 28 onm: 50880 M "1 cm “1 , MW monomer: 28.33kDa; for J591 scFv E 280 nm: 50880 M "1 cm “1 , MW monomer: 27.70kDa; for huJ591scFv E 28 onm: 50880 M "1 cm “1 , MW monomer: 27.28kDa; for 6C7.1 E 280 nm: 48610 M “1 cm “1 , MW monomer: 28.78kDa; and for 6C7.1 C E 280 nm: 48610 M “1 cm “1 , MW monomer: 28.89kDa. Aliquots were stored at -80°C.
  • scFv protein fragments Purity of the scFv protein fragments was assessed by SDS-PAGE/Coomassie brilliant blue staining, analytical size exclusion HPLC (BioSep SEC-S2000, Phenomenex) and a Western blot. Proteins were separated using NuPAGE 12% gels and MOPS buffer (Life Technologies). Gels were either stained with Coomassie brilliant blue or proteins were transferred to nitrocellulose membranes for subsequent Western blot detection using antiPentaHis (Qiagen) as primary antibody, Gam:HRP (Millipore) as secondary antibody and SigmaFastDAB (SigmaAldrich) as HRP substrate. Size exclusion HPLC was performed with PBS, pH 7.4 as mobile phase with a flow rate of 1 ml/min.
  • J591 scFv the labelling sequence, RAAALEHHHHHH, has a pi of 7.21 , and thus the proteins comprising this sequence did not constitute biomolecular complexes of the present invention.
  • the J591 scFv control has an Arg 6 amino acids away from the His residues.
  • An Arg in close proximity to His residues has shown a strong influence on the labelling efficiencies and therefore to compensate for the presence of an Arg in the J591 scFv control, an alternative huJ591scFv was synthesised with a labelling sequence containing no Arg residues.
  • the huJ591 scFv has a labelling sequence KLAAALEHHHHHH with a pi of 7.21 .
  • the 6C7.1 and 6C7.1 -Cys scFvs have identical amino acid composition to each other except for an additional Cys residue at the C- terminal of the His-Tag in the 6C7.1 C scFv.
  • the labelling sequences for 6C7.1 and 6C7.1 - Cys are TAAALEHHHHHH and TAAALEHHHHHHC respectively and the pi is 6.53 for both. Full details of the various proteins produced are set out in Table 3, in which all labelling sequences have been highlighted in bold.
  • the scFv proteins were produced in HEK239T cells with yields of purified protein (purity >95%) of 2-6mg/L culture supernatant.
  • the production and purification of the J591 scFvJWT and J591 scFv proteins were recorded by SDS PAGE and Western blot on nitrocellulose membrane and are displayed in Figure 24a and 24b respectively.
  • the J591 scFvJWT ( Figure 24a) appears as a monomer of 28kDa and can be seen in every sample added to the NuPage gel. Lanes A-C in the SDS-PAGE monitor the progress of extracting J591 scFvJWT from the culture supernatatant.
  • (His)6 recombinant proteins such as J591 scFvJWT have a high affinity and selectivity for the Ni-NTA and as a result, the majority of the protein impurities present in the supernatant, lane A, are discarded in the flowthrough of the Ni-NTA purification system.
  • Other protein impurities, principally BSA, unspecifically bound to the Ni- NTA are removed in the 35mM imidazole wash of the Ni-NTA column, lane B.
  • J591 scFvJWT was eluted from the column by competition with a 250mM imidazole solution, lane C. Further purification of the J591scFvJWT was required and was achieved with size exclusion chromatography.
  • J591 scFvJWT was eluted as a purified protein in the third peak, lane F, and concentrated to 1 .3mg/ml, lane G.
  • the corresponding Western blot reveals all the proteins present with a (His)6 tag which confirms the appearance of the J591 scFvJWT as a monomeric band at 28KDa on the SDS-PAGE.
  • the primary antibody marker used in the Western blot, antiPentaHis, is specific for (His)6 targeting.
  • FIG. 24b SDS-PAGE and Western Blot monitoring for the process of extracting and purifying the J591 scFv protein is shown in Figure 24b.
  • lane A-C represents the extraction of the protein from the culture supernatant and lane D-F represents the purification fractions from the SEC.
  • the J591 scFv appears as a monomeric protein at 27kDa and the purified (purity > 95%) sample can be seen in lane F concentrated to 1 .35mg/ml.
  • Confirmation for the presence of J591 scFv is given by the Western Blot which highlights the position of the (His)6 containing proteins on the SDS-PAGE.
  • All scFv protein fragments were radiolabeled by site-specific chelation of [ 99m Tc(CO) 3 ] + by the C-terminal (His)6-tag. To compare the relative labelling efficiencies, the proteins were radiolabeled at a range of 6 different concentrations in a 2:1 dilution series. Protein concentrations were calculated post addition of the [ 99m Tc(CO) 3 ] + radiolabelling solution. The highest concentration achieved for the protein in the [ 99m Tc(CO) 3 ] + radiolabelling solution was 28.2uM which for the J591 scFvJWT, J591 scFvJWT and huJ591 scFv proteins is equivalent to 0.8mg/ml.
  • the subsequent 5 protein concentrations in the dilution series were 14.1 uM, 7uM, 3.5uM, 1 .76uM and 0.88uM.
  • Concentrations of the purified 6C7.1 and 6C7.1 -Cys were not high enough to enable the protein concentration to reach 28.2uM in the [ 99m Tc(CO) 3 ] + labelling solution. Consequently, for 6C7.1 and 6C7.1 -Cys the highest protein concentrations achieved in the labelling reaction were 14.1 uM and 7uM respectively.
  • the J591 scFvJWT, J591 scFv, huJ591 scFv, 6C7.1 and 6C7.1 -Cys, proteins were labelled with [ 99m Tc(CO) 3 ] + via the C-terminal (His)6-tag for the comparative radiolabelling studies.
  • Preparation of the [ 99m Tc(CO) 3 ] + was achieved using the conventional IsoLink kits kindly provided by Covidien.
  • the Isolink kit was reconsitutued with 2200-2500MBq of 99m Tc0 4 " ( 99m Tc pertechnetate) in 400ul of saline and heated to 97°C for 30min.
  • the kit was neutralised with 1 M HCI to pH 7.5 (approximately 160ul) and quality control performed by thin layer chromatrographjy (TLC) to verify the conversion rate of 99m Tc0 4 " to [ 99m Tc(CO) 3 ] + .
  • TLC thin layer chromatrographjy
  • Glass backed silica gel 60 (Merck) TLC plates were used with a mobile phase of 1 % HCI in methanol for the quality control.
  • ScFv proteins in PBS at pH 7.4 were prepared to a concentration of: 42uM (1 .2mg/ml) for J591 scFvJWT, J591 scFv and huJ591 scFv; 21 uM (0.6mg/ml) for 6C7.1 ; and 10.5uM (0.3mg/ml) for 6C7.1 -Cys. Protein concentrations were determined by UV absorption at 280nm using a Nanodrop spectrophotometer.
  • Equation 1 Calculating percentage radiochemical yield from the TLC data recorded by the gamma counter.
  • Radiochemical yield (%) Total cpm from TLC strip X 100
  • J591scFv, huJ591 , 6C7.1 and 6C7.1 -Cys have lower labelling efficiencies and are similar to each other according to the graphs in Figure 26.
  • a slight increase in the radiochemical yield was observed for the J591 scFv. This is probably due to the labelling sequence of J591 scFv that contains an Arg residue 6 amino acids away from the (His)6.
  • the Arg amino acid has been replaced by a Lys or Leu residue and these proteins displayed the lowest radiochemical yield at all time points.
  • J591 scFvJWT exceeds the efficiency shown by the other proteins with standard (His)6 sequences. From Table 5 it is clear that J591 scFvJWT achieved a radiolabelling efficiency greater than 95% at the lowest protein concentration, 7uM, after incubation for 90minutes. For the non Arg/His containing proteins, the lowest protein concentration at which a radiochemical yield greater than 95% was achieved was 28.2uM after 90 minutes, J591 scFv and huJ591 scFv. This reveals that an Arg/His Tag has demonstrated an identical radiolabelling efficiency to that of a generic (His)6 tag with a significant 4 fold decrease in protein concentration at 90 minutes.
  • the experimental conditions described herein may be used as a guide to suitable conditions that may be employed in methods of manufacturing and radiolabelling the biomolecular complexes of the invention.
  • biomolecular complexes of the invention provide a number of important advantages as compared to comparators incorporating labelling sequences known from the prior art. These advantages (which include the ability to use lower concentrations and/or shorter incubation times, and to achieve higher levels of labelling) have significant utility in clinical, diagnostic and research settings.
  • J591 scFvJWT was radiolabelled with [ 99m Tc(CO) 3 ] + as previously described (B2.3). Once the radiochemical yield had reached 95%, the [ 99m Tc(CO) 3 ] + -J591 scFvJWT conjugate was incubated in a 1 :1 (v/v) ratio with fresh human serum at 37°C. Aliquots were taken at 0, 15, 30, 60, 120 and 240 minutes for TLC analysis using ITLC-SA chromatography paper and a mobile phase of 0.1 M citrate buffer at pH 5. In addition, samples were obtained at the same time points and immediately frozen in liquid nitrogen.
  • an SDS-PAGE was carried out on all the samples collected at the 0, 15, 30, 60, 120 and 240 minute time points.
  • control samples were included on the SDS-PAGE gel as references for the individual components: [ 99m Tc(CO) 3 ] + , serum proteins and J591 scFvJWT.
  • the control samples were [ 99m Tc(CO) 3 ] + only, [ 99m Tc(CO) 3 ] + -serum protein conjugates and [ 99m Tc(CO) 3 ] + - J591 scFvJWT conjugate.
  • the results of the SDS-PAGE can be seen in Figure 29 with the Coomassie staining image on the left (A) and autoradiograph on the right (B).
  • Lanes C-H in the autoradiograph confirm the presence of the radiolabeled J591 scFvJWT as single black bands at 28kDa corresponding to the monomeric protein.
  • Lane A is a control and in the autoradiograph the black band corresponds to [ 99m Tc(CO) 3 ] + conjugated to serum proteins.
  • it is not possible to observe any radioactivity which is understandable as the stable loaded in this row was [ 99m Tc(CO) 3 ] + which is small and highly charged. It is very likely that it has travelled to the end of the NuPAGE gel and is no longer registered on the SDS-PAGE.
  • the corresponding Coomassie blue stained image ( Figure 29, A), identifies the location of the serum proteins within the serum containing samples.
  • J591 scFvJWT is stable in serum for at least 4 hours at 37 °C.
  • the other four proteins (J591 scFv, huJ591 scFv, 6C7.1 and 6C7.1 -Cys) have previously been analysed for serum stability by Dr Florian Kampmeier and they demonstrate an identical behaviour.

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Abstract

La présente invention concerne des complexes biomoléculaires qui sont utiles dans le marquage de protéines à l'aide de groupes métal tricarbonyle. Le complexe biomoléculaire selon l'invention comprend une protéine d'intérêt et une séquence de marquage susceptible d'être conjuguée à un métal tricarbonyle ([M(CO)3]+). La séquence de marquage est constituée d'entre 6 et 15 résidus acides aminés, parmi lesquels entre 4 et 6 résidus correspondent à des résidus histidine et parmi lesquels au moins 2 résidus sont choisis dans le groupe constitué par des résidus lysine et/ou arginine. La séquence de marquage présente un point isoélectrique (pI) supérieur ou égal à 9. Les complexes biomoléculaires de l'invention sont utiles dans l'imagerie moléculaire, dans des applications diagnostiques et thérapeutiques. L'invention concerne également des molécules d'acide nucléique codant pour de tels complexes biomoléculaires ; des procédés de production de tels complexes biomoléculaires ; des complexes biomoléculaires destinés à être utilisés comme médicaments en radiothérapie ; et des méthodes de traitement, de diagnostic et d'imagerie moléculaire utilisant de tels complexes moléculaires.
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